Induced pluripotent stem cell-derived human hepatocyte-like cells (iHLCs) can be used to study inter-individual variability in hepatitis C (HCV disease progression and effects of multiple drug regimens. These iHLCs can also mitigate the sourcing limitation with primary human hepatocytes (PHH) for such investigations. Others have shown that iHLCs in conventional confluent monolayers can be infected with live HCV and can sustain viral replication for ~2 weeks. However, the infection efficiency reported is fairly low (~-3%), potentially because the cells need to be further differentiated towards the adult liver phenotype. Indeed, the differentiation status of iHLCs has been shown to correlate strongly with permissiveness of these cells to HCV infection. We have recently adapted our previously developed micropatterned co-culture (MPCC) technique with key modifications to iHLCs (iMPCCs). We found that iMPCCs are more functionally mature via several functional metrics than conventional cultures of iHLCs from the same donors, and more mature than the starting material differentiated using scaled-up but typical protocols by our collaborator. Additionally, iMPCCs sustain liver functions for 1 month in vitro. Here, we hypothesize that the iMPCC system containing better-differentiated iHLCs (including better polarity due to precisely defined architecture) will support HCV infection with higher efficiencies than conventional cultures of IHLCs and sustain HCV replication and de novo viral production for 3- 4 weeks in vitro. In particular, we will assess long-term expression of HCV entry receptors (i.e. CD81) in iMPCCs, entry of HCV pseudo-particles, and the infection efficiency using live virus (cell culture adapted and from patient sera). We will compare the data in iHLCs to stable PHHs in the same MPCC configuration. We anticipate that due to the phenotypic stability of our culture model, we will be able to monitor infection processes longitudinally. In the future, we plan to generate iHLCs from different patients with specific HCV progression profiles, investigate mechanisms by which iMPCCs improve HCV permissiveness of iHLCs, and study in iMPCCs the interaction of iHLCs with liver stromal cells that are known to modulate HCV infection.